Reference to any prior art in this specification is not, and should not be taken as, an acknowledgement or any form of suggestion that this prior art forms part of the common general knowledge in Malaysia, Australia or any other country.
Bibliographic details of the publications referred to by author in this specification are collected at the end of the description.
The demand for oil and fats is expected to increase dramatically with the increase in world population. Oil palm (Elaeis guineensis and Elaeis oleifera), which produces the palm oil and palm kernel oil, is the highest yielding oil crop in the world and was forecasted to contribute around a quarter of the world's oil and fats demand by the year 2020 (Rajanaidu and Jalani, 1995). Due to the demand, there is a need to increase the quality and yield of palm oil and palm kernel oil and to rapidly develop new characteristics when required.
Current methods of improvement via conventional breeding are very slow due to the nature of the breeding process and the very long generation time for oil palm plants. Genetic engineering offers a means of manipulating the characteristics of plants. However, until the advent of the present invention, protocols for its use in oil palm plants were not well developed.
Genetic engineering is a specialized method of improving plant quality by introducing foreign genes into the whole plant by genetic transformation. Genetic engineering processes are often unique to particular plants. An efficient tissue culture system is required in order to produce complete chimeric-free transgenic plants after successful delivery and integration of foreign genes into cells that are able to regenerate into a whole plant. To engineer any plant genetically, it is important to obtain a transgenic plant that possesses the gene(s) in the whole plant. The gene(s) need to be expressed in the whole plant if the objective is, for example, to develop disease or herbicide resistant plants. On the other hand, if the interest is in improving or modifying a specific trait in a particular tissue such as in fruit, tissue specific promoters are desirable. In both situations, the presence of chimeric plants is undesirable.
The genetic transformation process involves the uptake of a foreign gene by competent cells, its integration into the chromosome and subsequently the expression of the genes product. The process starts with the penetration of a gene into a cell through the cell wall. Various methods are available for plant gene transfer and are basically divided into two main groups; namely Agrobacterium-mediated gene transfer and direct gene transfer. Discussion on some of the various plant transformation methods available is provided below. However, none of the methodologies has been successful in producing chimeric-free transgenic oil palm plants.
Agrobacterium-Mediated Transformation
Agrobacterium tumafaciens is capable of introducing foreign genes into plant cells via a tumor-inducing plasmid known as Ti. One region of Ti, known as T-DNA (transferred DNA), contains genes which are not involved in the transfer process, making it possible to replace those genes with genes of interest for genetic engineering purposes. By a mechanism which remains unknown, although thought to be analogous to bacterial conjugation, the T-DNA is transferred into the plant cell and is stably inserted into the nuclear DNA in a process thought to involve proteins coded by the Vir E gene.
Expression and stable delivery of foreign genes into plants by Agrobacterium-mediated gene transfer has been demonstrated (Bevan et al., 1983; Fraley et al., 1983; Zambryski et al., 1983; Horsh et al., 1985). Since then, dicots have been identified as being the most efficient host for Agrobacterium-mediated transformation. Even though monocots are not the natural host of Agrobacterium (DeClene and Deley, 1976), there is now increasing evidence that, under certain conditions, Agrobacterium can be used to transform monocots (Bytebier et al., 1987; May et al., 1995; Ishida et al., 1996; Rashid et al., 1996).
Protoplast-Mediated Transformation
Protoplasts are plant cells whose cell wall has been removed by enzymes such as cellulase, pectolyase and macerozyme. These naked (plasma membrane) cells are surrounded by tiny pores, which are nevertheless too small for direct penetration of naked DNA (genes). However, the use of polyethylene glycol (PEG) or a short high-voltage electric pulse (electroporation) can produce transient pores, which allow DNA to enter the cells (Lorz et al., 1985). Transgenic monocot plants, produced using protoplasts have been reported (Horn et al., 1988; Rhodes et al., 1988; Shimamoto et al., 1989; Wang et al., 1992). The most significant disadvantage of using protoplast-mediated transformation is that the regeneration of whole plants from monocot protoplasts is very difficult (Potrykus, 1990).
Microprojectile Bombardment or Biolistics Transformation
Biolistics involves the use of an apparatus that accelerates DNA-coated high density metal particles at a high velocity sufficient to penetrate plant cell walls and membranes (Klein et al., 1987). The metal particles of about one to two microns in diameter are coated with a gene of interest prior to bombardment in the presence of Ca+ ions and spermidine. Small puncture holes produced on the cell walls and membranes after particle penetration will close spontaneously. This method has been the method of choice for monocots and most of the species which have failed to be transformed using Agrobacterium-mediated transfer or protoplast transformation have been successfully transformed using this method. The most commonly used tissue for biolistic-mediated transformation is derived from embryogenic suspension cultures (Fromm et al., 1990) and embryogenic callus cultures (Bower and Birch, 1992; Vasil et al., 1992). However, it was considered that transformation using primary explants with high regeneration capacity was superior (i.e. scutellar tissue can reduce the time required to produce transgenic plants and also reduce the risk of somaclonal variation (Christou et al., 1991; Jahne et al., 1995). Other tissues used for transformation and production of transgenic plants are protocorm (Kuehnle and Sugii, 1992) immature inflorescence (Barcelo et al., 1994), microspore (Jahne et al., 1994) and shoot meristem (Lowe et al., 1995) tissues. The only disadvantage of microprojectile bombardment is the low efficiency of obtaining stable transformation in comparison with transient expression.
Tissue Electroporation Transformation
Tissue electroporation has been used to transfer DNA into enzymatically or mechanically wounded tissue. Stably transformed maize type-1 callus and transgenic plants have been obtained using this method (D'Halluin et al., 1992). Other tissues that have been used for transformation are scutellum (Kloti et al., 1993), bisected mature embryos (Xu and Li, 1994), suspension culture cells (Laursen et al., 1994), embryogenic calli (Arencibia et al., 1995) and mechanically wounded immature embryo (Xiayi et al., 1996).
Silicon Carbide Transformation
This method involves mixing (e.g. by vortexing) cells in a solution containing whiskers (silicon carbide) and plasmid DNA. Collision between cells and whiskers results in cell penetration and delivery of DNA (Kaeppler et al., 1990). Fertile transgenic maize plants have been reported using suspension cultured cells (Frame et al., 1994).
Microinjection Transformation
Microinjection requires microcapillaries and microscopic equipment to deliver DNA directly into the nucleus. Injection results in a micro-hole on the cell wall, thought to have no effect on viability. This method has only been reported successfully in dicots (Schnorf et al., 1991; Bechtold et al., 1993). No success has yet been reported or confirmed in a monocot. The disadvantage of this method is that only one cell can be transferred per injection, making it a time consuming procedure.
Laser Microbeam Transformation
A laser microbeam can be used to overcome the cell wall barrier of cells, by punching a small hole on the surface via focusing the beam within the cell. This allows the manipulation of the nucleus or organelles without opening the cell membrane (Weber et al., 1990). The hole facilitates the entrance of foreign DNA into the cell and closes within 1-2 seconds (Weber et al., 1989). Production of transgenic plants has been reported (Guo et al., 1995). However, only GUS staining was used to prove that integration of the transgene into the regenerated plants had occurred. No Southern blot hybridization or progeny test data were shown in the report.
Imbibition of Seeds Transformation
The incubation of dry seeds or mature embryos in DNA solution has been shown to result in transformation (Topfer et al., 1989). This procedure is based on osmotic pressure differences forcing DNA into the seeds. However, no evidence of stable integration was provided.
DNA Integration
Transgene(s) needs to be stably integrated into the plant genome and subsequently expressed. The presence of the transgene can be confirmed by Southern blot hybridization (Sambrook et al., 1989) or polymerase chain reaction (PCR) (Chee et al., 1991). The presence of the transgene in the high molecular weigh undigested DNA as shown by Southern hybridization, is routinely used to confirm the total integration of the transgene (Casas et al., 1995). PCR is useful for screening a large quantity of samples but cannot be used to demonstrate the integration of the transgene.
Gene Expression (Protein)
Once transgene integration has been confirmed, it is important to determine whether the transgene is functional (expressed) in the transgenic plants. Method that can facilitate early detection of expression is the ability to detect survival in the presence of a selection agent, such as an antibiotic or a herbicide. The GUS reporter gene also can be used, together with a histochemical or fluorimetric assay, to examine gene expression early (Jefferson, 1987). Enzymatic assay for the detection of bar gene expression by acetylation of PPT in the presence of acetyl-CoA (DeBlock et al., 1987) can also facilitate early detection of transgene expression. Similar assays have been used for detection of hmr gene expression (Datta et al., 1990).